Diagnostic composition



United States Patent Ofifice 3,642,496 Patented July 3, 1962 Thisinvention relates to a new and improved diagnostic composition and isparticularly concerned with a diagnostic test which is useful for thequalitative detection and quantitative determination of glucose inbiological fluids, such as urine, and wherein the reagent composition isincorporated upon a bibulous carrier.

The detection of glucose in urine as well as the determination of itsconcentration therein is of great importance for diabetic patients whomust control their diets so as to regulate their sugar intake and whomust frequently be guided in this regard by a regular check on urineglucose. But beyond its usefulness in regular urine testing on knowndiabetics by both patients and physicians, this glucose indicator mayalso be used efiiciently in routine urinalyses in hospitals andphysicians oflices, in diabetes detection screening programs, in thedifierentiation of glucosuria from other meliturias, and the like.

Because early diagnosis and continued control are so important indiabetes, a urine-sugar test, to be of greatest value, must beconveniently rapid, simple enough for any patient to learn with ease,accurate enough to serve the clinician, and sensitive enough to reflectvariations in the patients condition. Moreover, the reagent compositionmust be adequately stable.

Procedures for the detection of sugar in urine are well known inclinical chemistry. One such procedure utilizes Benedicts copperreduction test, another employs a selfheating alkaline coppe reductiontest in tablet form (US. Patent No. 2,387,244), still another involves atest which depends solely on the action of enzymes (U.S. application No.514,395, filed June 9, 1955, by Alfred H. Free and assigned to theassignee of the present invention). None of these procedures,'however,has entirely satisfied the above-mentioned requirements.

We have now found a novel and highly useful glucosedetecting means whichrepresents an important improvement and a fresh approach to the problemof determining glucose in various materials including body fluids, suchas urine, by a technique which utilizes a diagnostic composition thatsurpasses even the latest and most commonly used glucose tests instability and quantitation.

Specifically, we have now found that metalloporphyrins, which are notoperable per so, are, however, when activated by certain nitrogenoussubstances, surprisingly and unexpectedly capable of acting as catalyststo induce the oxidation of indicators by hydrogen peroxide formed whenglucose is aerobically oxidized in the presence of glucose oxidase. Theprinciples underlying these reactions are well known and need not beexpounded.

A diagnostic composition according to the present invention comprises asessential constituents glucose oxidase, a metalloporphyrin, such ashemin, complex-forming compounds containing amino-donor groups, such asZ-aminobenzothiazole, pyridine, bipyridyl, bipyridylpyridine, nicotinicacid or the like, and color-forming substances or indicators, such as2,7-diaminofluorene, o-tolidine, leucoindophenols, etc.

The metalloporphyrin used in the preferred embodiments of this inventionis hemin, which is commercially available and whose full chemical nameis 1,3,5,8-tetramethyl-2,4-diviny1porphine-6,7-dipropionic acidferrichloride, (C H N O FeCl). The following structural formulacharacterizes its complex chemical nature:

H3C CH=CH n 1 CH3 l /N Fe 4% nooccn cn 2 Haw H nooccn ca CH3 Hernin andthe other 'metalloporphyrins that can be 0 employed in accordance withthe inventive concept react wherein the 4-membered ring represents anoctahedral porphyrin ring system, M represents Fe, Co, Ni, Mn, Mg,

stands for a nitrogen atom of a complex-forming aminodonor group.

In place of a metalloporphyrin it is also contemplated to employ otherequivalent metallic complexes or chelates, such as phthalocyanines, thestructure of which is closely analogous to that of the porphine nucleuspresent in hemin. Further catalytic materials useful in the practice ofthis invention are ferrocene compounds, the ferrous salt ofdimethylglyoxime and the like.

The diagnostic composition of our invention is prepared, due toincompatibility of ingredients, by way of two separate solutions, onecomprising the glucose oxidase and the indicator substance dissolved in1:1 alcohol-water and the other containing hemin and the nitrogenoussubstance in an alcoholic medium.

It is understood that such additives as suitable protective agents andwetting agents may be incorporated therein. Furthermore, this glucosetest may contain a suitable inert dye to impart to the composition auniform background color as well as an appropriate buffer system tomaintain a desired pH range (pH 3 to 7 with a pH of 5.4 beingpreferred).

The following examples illustrate in greater detail a number ofembodiments of our invention as well as their method of manufacture.These examples demonstrate the flexibility of our invention and thevarious modifications possible within the purview of the inventiveconcept and should therefore not be construed as limiting the ambit ofthe appended claims.

EXAMPLE I 0 of water until a clear solution resulted. Then 20 ml. of

a pH 5.4 buffer solution (prepared by dissolving 352.8 g. of sodiumcitrate monohydrate and 25.6 g. of anhydrous citric acid in water anddiluting to 1000 ml.) was added with stirring. A solution of 2 g. ofgelatin and 150 mg. of ED. & C. Red No. 3 (disodium salt of9-o-carboxyphenyl 6 hydroxy 2,4,5,7 tetraiodo-3-isoxanthone) in 100 ml.of hot water was prepared and 20 ml. of this solution was added to thealgin-bufier solution with stirring. Then a solution of 200 mg. of2,7diamino-fiuorene dihydrochloride in 20 m1. of hot 50% ethanol wasadded. A solution of 400 mg. of glucose oxidase (16,000 units per gramwhich had been heated at 100 for hours to destroy catalase) in 5 ml. ofWater was added. The resulting solution was kept at 4045 by warming asnecessary.

Solution B.Hemin, 50 mg, and 500 mg. of 2-aminobenzothiazole were addedto 50 ml. of 95 ethanol and 50 ml. of 10% aqueous polyvinyl alcohol andthe mixture was heated to boiling and shaken for two minutes. The excesshemin was removed by filtration, and the filtrate was allowed to cool toroom temperature.

Preparation of Diagnostic Strip Bilbulous strips, such as filter papercut into narrow strips, small sticks of wood, or other porous orabsorbing material with a water-impervious barrier of ethyl cellulose,one-half inch from the tip, were dipped into 'solution A so that throughthe process of submersion Procedure of Testing In use, an impregnatedstrip made as described above is dipped into the liquid specimen to betested. When contacted with urine containing glucose, test strips gavepositive reactions in about one minute evidenced by various shades ofblue color as follows:

With urine containing 1% or 2% glucose an intense blue color developedin one minute, while with urine containing 0.1% glucose a faintly bluecolor developed in one minute. With urines containing 0.25% and 0.5%glucose the blue colors which developed in one minute were ofintermediate intensities, the urine containing the most glucose beingthe darkest blue. When dipped in urine containing no glucose, the sticksunderwent no color change. A simple color chart based on this phenomenonmay be conveniently prepared for use in estimating the glucose contentof the urine being tested.

Other specific embodiments of our invention are illustrated by thefollowing examples:

EXAMPLE II (A) Twelve ml. of a buffer solution (which was prepared bydissolving 7.4 g. of anhydrous citric acid and 32.6 g. of trisodiumcitrate dihydrate in 100 ml. of water) was added rapidly with goodstirring to 10 ml. of a warm gelatin solution (which was prepared bydissolving 4.8 g. of gelatin in 100 ml. of boiling water). One hundredmg. of o-tolidine dihydrochloride was mixed with 10 ml. of 95% ethanol(2B), and the mixture was added rapidly to the gelatin-buffer solutionwith good stirring. Then 10 ml. of a glucose oxid-ase solution wasadded, which was prepared by dissolving 0.5 g. of glucose oxidase(having an activity of 16,000 units per gram) in 50 ml. of water, andthe mixture was stirred until well mixed. Paper strips were dipped intothis solution and dried at 100 C. for 10 minutes.

(B) Five mg. of hemin was dissolved in two to ten drops of pyridine, andthe mixture was diluted with 95% ethanol. The strips were dipped intothis solution and then (While still wet) into urine containing 1%glucose.

EXAMPLE III A mixture of 50 mg. of hemin, 500 mg. of nicotinic acid, 50ml. of ethanol and 50 ml. of 10% aqueous polyvinyl alcohol was warmedand shaken for 5 minutes. The excess hemin was removed by filtration.Strips described in Example IIA were impregnated with this mixture anddried at for 10 minutes. These strips developed a blue-green color after5 minutes with urine containing 1% glucose and only a very faint greencolor with urine containing glucose.

EXAMPLE IV When 50 mg. of Z-aminobenzothiazole was substituted for thenicotinic acid in Example III, the strips were a little more active, anda blue-green color developed with 1% glucose in urine in 3 minutes.

EXAMPLE V (A) Twenty ml. of hot buffer solution (which was prepared bydissolving 352.8 g. of sodium citrate monohydrate and 25.6 g. ofanhydrous citric acid in water and diluting to 1000 ml.) was added withstirring to a solution of 200 mg. of algin in 20 ml. of water. Then 20ml. of a hot gelatin-dye solution (Which was prepared by dissolving 2 g.of gelatin and mg. of ED. & C. Red No. 3 dye in 100 ml. of water) wasadded with stirring and followed by a solution of 2,7-diaminofiuorenedihydrochloride (200 mg.) in 20 ml. of hot 50% aqueous ethanol. Finallya solution of 400 mg. of glucose oxidase (16,000 units per gram) in 5ml. of water was added with good stirring, and paper strips wereimpregnated at once with the warm mixture. The strips were dried at 100for 10 minutes.

(13) These strips were dipped into the hemin-nicotinic acid complexsolution as in Example II-I and dried at 100 for 10 minutes. When dippedin urine containing 1% glucose, a moderate blue color developed in 2minutes.

With glucose in urine only a faint color change was observed in 2minutes.

In summary, this invention pertains to a diagnostic composition for thedetection of glucose in body fluids,

and especially in urine, consisting of a bibulous strip or stick thathas been impregnated with a composition comprising glucose oxidase, ametalloporphyrin, such as hemin, a complex-forming compound containingaminodonor groups, such as 2-aminobenzothiazole, nicotinic acid orpyridine to activate the metalloporphyrin and a color-forming substance,such as 2,7-diaminolluorene or o-tolidine, which is oxidizable byhydrogen peroxide in the presence of the activated metalloporphyrin. Thecomposition containing glucose oxidase, hemin, Z-aminobenzothiazole and2,7-diaminofiuorene constitutes the preferred embodiment of thisinvention.

What is claimed is:

1. A diagnostic composition for detecting glucose which comprisesglucose oxidase, a metalloporphyrin, a complexforming compoundcontaining amino-donor groups selected from the class consisting of2-aminobenzothiazole, nicotinic acid and pyridine to activate saidmetalloporphyrin and a color-forming substance taken from the groupconsisting of 2,7-diaminofluorene and o-tolidine, oxidizable by hydrogenperoxide in the presence of the activated metalloporphyrin.

2. A diagnostic composition for detecting glucose which comprisesglucose oxidase, hemin, a complex-forming compound selected from theclass consisting of Z-aminobenzothiazole, nicotinic acid and pyridine toactivate said hemin and a color-forming substance taken from the groupconsisting of 2,7-diaminofiuorene and o-tolidine, oxidizable by hydrogenperoxide in the presence of the activated hemin.

3. A diagnostic composition for detecting glucose which comprisesglucose oxidase, hemin, Z-aminobenzothiazole to activate said hemin byforming a complex therewith and 2,7-diaminofluorene oxidizable byhydrogen peroxide in the presence of the activated hemin.

4. A glucose-detecting means including a bibulous carrier impregnatedwith a diagnostic composition comprising glucose oxidase, hemin,Z-aminobenzothiazole to activate said hemin, and 2,7-dia-minofluoreneoxidizable by hydrogen peroxide in the presence of the activated hemin.

5. A glucose-detecting means including a bibulous carrier impregnatedwith a diagnostic composition comprising glucose oxidase, hemin, anamino-donor selected from the group consisting of Z-aminobenzothiazole,nicotinic acid and pyridine to activate said hemin and2,7-diaminofluorene oxidizable by hydrogen peroxide in the presence ofthe activated hemin.

rier impregnated with a diagnostic composition comprising glucoseoxidase, hemin, pyridine to activate said hemin and 2,7-diaminofiuoreneoxidizable by hydrogen peroxide in the presence of the activated hemin.

References Cited in the file of this patent FOREIGN PATENTS AustraliaSept. 27, 1956 OTHER REFERENCES Fearon, W. B.: Introduction toBiochemistry, 1947, published by Grunt Stratton, N.Y., pp. 212, 251,459.

Sumner, J. B., and Somers, G. F.: Chemistry and Methods of Enzymes,1953, Academic Press, N.Y., pp. 225-226.

1. A DIAGNOSTIC COMPOSITION FOR DETECTING GLUCOSE WHICH COMPRISESGLUCOSE OXIDASE, A METALLOPORPHYRIN, A COMPLEXFORMING COMPOUNDCONTAINING AMINO-DONOR GROUPS SELELCTED FROM THE CLASS CONSISTING OF2-AMINOBENZOTHIAZOLE, NICOTINIIC ACID AND PYRIDINE TO ACTIVATE SAIDMETALLOPORPHYRIN AND A COLOR-FORMING SUBSTANCE TAKEN FROM THE GROUPCONSISTING OF 2,7-DIAMINOFLUORENE ANND 0-TOLIDINE, OXIDIZABLE BYHYDROGEN PEROXOXIIDE IN THE PRESENCE OF THE ACTIVATED METALLOPORPHYRIN.